The present invention relates generally to the field of annexins. More particularly, it relates to compositions and methods for treating and diagnosing a subject by delivering compounds to a specified target using annexins, variants of annexins, and derivatives thereof.
Pharmacological and genetic treatments of diseases are based on the delivery of pharmacologically active compounds to diseased cells where the compounds act preferably intracellularly. Current therapeutic treatments employ systemic delivery of a drug, where the drug circulates through the entire body before reaching its desired target. This method of drug delivery results in systemic dilution of the compound. As a result, a patient requires higher concentrations of the drug to achieve the favored therapeutic efficacy. Higher drug concentrations naturally increase any undesired toxic side-effects of the pharmacologically active compounds. In addition, higher concentrations generally increase the cost of any drug.
To circumvent these problems regional drug delivery systems have been designed ranging from local applications to drug targeting. Regional delivery, however, is often insufficient because it does not ensure delivery into a targeted cell. In order to act efficiently, most drugs, such as chemotherapeutics and photodynamic agents, must enter a diseased cell. Several systems have been described that may be used to deliver compounds to a cell.
Cell-Penetrating Peptides or Protein Transduction Domains
Cell-Penetrating Peptides (“CPP”) or Protein Transduction Domains (“PTD”) form a class of peptides that are able to cross the plasma membranes of eucaryotic cells, (Lindgren, et al., Trends Pharmacol. Sci. 21:99-103 (2000)). Most CPP/PTDs are derived from larger proteins of viral origin. CPP/PTDs can also be engineered by design (U.S. Pat. No. 6,495,663). The mechanism by which CPP/PTDs translocate over the membrane is not entirely understood but it appears to be energy-independent. The potential use of CPP/PTDs in drug delivery has been recognized and examples of conjugates between CPP/PTDs and cargo such as small chemical entities, peptides, and proteins have been published (Schwarze, et al., Trends Pharmacol. Sci. 21:45-48 (2000); U.S. Pat. No. 6,472,507). The drawback of CPP/PTDs is their lytic activity at high concentrations (Scheller, et al., J. Pept. Sci. 5:185-94 (1999)). Additionally, CPP/PTDs do not have a targeting capability and do not discriminate between healthy and diseased cells. Hence, the toxic side-effects of pharmacological compounds are not diminished by their conjugation to CPP/PTDs. Rather, complexes formed from CPP or PTD and a pharmacological compound will enter both healthy and diseased cells. Targeting functionality has been attempted by conjugating CPP/PTDs to antibodies specific for antigens in tumors. The disadvantage of this approach, however, is that the targeting function of the antibody deteriorates as a result of the conjugation to the CPP/PTDs (Niesner, et al., Bioconjug Chem. 13:729-36 (2002)).
Ligands for Plasma Membrane Receptors.
Several pinocytic pathways have been described through which cells are able to internalize compounds from the environment. One of the pinocytic pathways comprises the receptor-ligand mediated endocytosis (Conner, et al., Nature 422:37-44 (2003)). When the ligand binds to the receptor, the whole complex is internalized in small intracellular vesicles, which will travel through the cell depending on the receptor-ligand complex involved. This pathway can be employed to bring drugs into a cell through coupling the drug or the drug transporting vehicle such as liposomes to a ligand specific for surface receptors that show endocytosis upon ligation. Examples of such ligands that induce internalization are ligands for the hFGF-receptor (U.S. Pat. No. 6,551,618), ligands for the asialoglycoprotein receptor (U.S. Pat. No. 5,885,968), and ligands for the transferring receptor (U.S. Pat. Nos. 6,511,967, 5,154,924, Qian, et al., Pharmacol. Rev. 54:561-87 (2002)). This type of internalization does not, in most cases, achieve an optimal targeting function for drug delivery. Moreover, recent data show that internalization via the receptor-ligand pathway may suppress the pharmacological action of the internalized drug (Sato, et al., Pharm. Res. 19:1736-44 (2002)).
One compound that can be used as a targeting agent and capturing agent is Annexin A5. In order to detect Annexin A5 bound to the surface, it has been conjugated to reporter compounds. In the past, Annexin A5 conjugates have been prepared by reacting reporter compounds with amino and hydroxyl groups present in the Annexin A5 molecule. Annexin A5, however, has more than one amino group and more than one hydroxyl group. As a result, chemical coupling with these functional groups yields mixtures of different stoichiometric complexes of Annexin A5 and the reporter compound. The outcome of these coupling procedures is random and contains complexes that lack the ability to bind to aminophospholipids because a set of the reacting amino acids are involved in phospholipid binding. These problems impair the quality of the Annexin A5 conjugates. In order to improve quality, the mixture of Annexin A5 conjugate has been separated by additional purification techniques.
In an attempt to improve Annexin A5 for conjugation, the single sulfhydryl group in Annexin A5, which is provided by a cysteine residue at position 315, has been coupled with a single chain urokinase in an attempt to remedy the problems with the amino and hydroxyl groups discussed above (U.S. Pat. No. 5,632,986). The complexes of Annexin A5 and urokinases target the urokinases to intravascular sites in order to the increase local fibrinolytic activity. The sulfhydryl group, however, is buried inside the molecule and urea induced unfolding of Annexin A5 is required to expose the sulfhydryl group to the surface of the protein. This procedure is used to allow conjugation to occur. Subsequent refolding of the Annexin A5, however, is required to regain the phospholipid binding capabilities of Annexin A5. Refolding procedures are not one hundred percent effective and typically generate an ineffective mixture of binding and non-binding annexin molecules.
U.S. Pat. No. 5,632,986 also describes the chemical introduction of multiple sulfhydryl groups with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”) into the Annexin A5 molecule for conjugation purposes. This procedure generates the same problems experienced with the procedure employed for the amino and hydroxyl groups discussed above. U.S. Pat. No. 5,632,986 further describes a variant of Annexin A5 with an extended N-terminus of ten amino acids comprising one cysteine residue in order to allow conjugation to a reporter compound. These variants have 329 amino acids of which two are cysteines. These variants may pose the problem of forming intramolecular disulfide bridges, thereby impairing the Annexin A5's ability to bind to phospholipids.
Bioactive compounds have also been conjugated to Annexin A5 by recombinant preparations of chimeras of Annexin A5 and the bioactive compounds (Tait, et al., Journal of Biological Chemistry 270:21594-99 (1995), U.S. Pat. Nos. 5,632,986). 6,323,313 describes variants of Annexin A5 with the extension of N-terminus part of Annexin A5 with an extra set of amino acids of which one is a cysteine residue. The extra amino acids with the cysteine residue are introduced with the purpose to chelate spontaneously the 99mTc radionuclide thereby obliterating the need for conjugation chemistry in order to radiolabel Annexin A5. This is only possible for compounds that show spontaneous and non-covalent attachment to the sulfhydryl groups. The problem with this type of interaction is the ability of the chelate reaction to reverse, i.e., the attached compound can dissociate from the Annexin A5 variant. The rate of dissociation depends on several factors, such as the compound and the amino acids capturing the compound.